Oligopeptide aldehydes useful as specific inhibitors of enterokinase

ABSTRACT

Compounds of the general formula: ##STR1## wherein n is an integer from 2 to 6, m is an integer from 2 to 4, p is an integer from 1 to 3, R is H, an amino protecting group conventionally used in peptide chemistry or a solid phase support, R 1  is H or a carboxy protecting group conventionally used in peptide chemistry and R 2  is H or alkyl or H 2  N--C═NH are useful for inhibiting the activity of enterokinases.

This invention relates to peptides and is particularly concerned withnew oligopeptides having selective inhibitory activity againstenterokinase.

Acute necrotising pancreatitis results from runaway activation ofdigestive enzyme precursors within the pancreas itself. We have shownthat the disease is initiated by the displacement of enterokinase fromthe proximal intestine into the circulation, its carbohydrate dependentuptake by the liver, transfer in catalytically active form to bile theentry of bile-bourne active enterokinase into the pancreatic ducts.

We have now prepared oligopeptide aldehydes which exhibit selectiveinhibitory activity against the proteinase enterokinase (EC 3.4.21.9)but not against other proteinases of the type inhibited by leupeptin andantipain.

Accordingly the present invention provides a compound of the generalformula: ##STR2## wherein n is an integer from 2 to 6, m is an integerfrom 2 to 4, p is an integer from 1 to 3, R is H, an amino protectinggroup conventionally used in peptide chemistry or a solid phase support,R₁ is H or a carboxy protecting group conventionally used in peptidechemistry and R₂ is H or alkyl or ##STR3##

In the compound of formula I, it is preferred that m be 3 and R₂ be##STR4## so that the terminal amino aldehyde residue of the oligopeptideis argininal. Further compounds of interest are those in which m is 4and R₂ is H or a C₁ -C₄ alkyl group e.g. methyl, so that the terminalamino aldehyde residue is lysinal or an N-alkyl lysinal. Higher or lowerhomologues of argininal or lysinal are also of interest in which thetrimethylene or tetramethylene group is replaced by monomethylene ordimethylene.

It is also preferred that p represent 1 so that there is at least oneaspartic acid residue present in the block of amino acids linked to theamino aldehyde. There will be at least two amino acid units directlybonded to the terminal amino aldehyde unit. These amino acid units willnormally be the same amino acid unit but need not necessarily be thesame. For example, although aspartic acid units are preferred, one ormore may be replaced for example glutamic acid.

n will be an integer of at least 2 up to 6 and it is preferred that n be2 or 4 since we have found that the selective inhibitory action of theseoligopeptides is at its greatest for the tripeptide and pentapeptide.

It is also preferred that the C-terminal amino aldehyde have Lconfiguration at the α-carbon atom but L configuration is not essentialat the other α-carbon atoms in the oligopeptide. D configuration mayconfer resistance to degradation in vivo.

It is also preferred that the terminal amino aldehyde be L-argininal andthat the amino dicarboxylic acids linked to it all be aspartic acid sothat the oligopeptide is a polyaspartyl argininal.

The compounds of major interest are the compounds in which the γ-carboxygroup in the amino dicarboxylic acid units is unprotected, that is tosay R₁ is H, as the carboxy protecting groups must be removed tomaximise the inhibitory activity. The inhibitory activity is influencedby the presence or absence of the amino protecting group R so that R canbe H, a protecting group in the active compounds or a solid phasesupport. The invention extends to the protected as well as theunprotected forms of the oligopeptides as, as a result of the syntheticmethods employed, the oligopeptides are normally first obtained inprotected form and the protecting groups are removed in a final stage ofsynthesis in accordance with conventional methods used in peptidechemistry.

When the terminal amino group is to be protected, it is protected withany of the protecting groups conventionally used, for example tertiarybutyloxy carbonyl, benzyloxy carbonyl or acetyl. These groups are ofinterest primarily because of their stability during the reactionconditions conventionally encountered in peptide synthesis and theirease of removal under acid conditions. However, other amino protectinggroups can be used, for example other acyl protecting groups such assimple carboxylic acyl groups or more complex carboxylic acyl groupsincluding oligopeptide residues which arise from peptide synthesis.Solid phase supports for the substitution of the α-NH₂ terminus mayinclude polysaccharide based matrices such as Agarose or Dextran orpolyacrylamide based matrices.

Similarly, the ω-carboxy group of the amino dicarboxylic acid unitsrequire protection during peptide synthesis and again, use may be madeof any of the protecting groups conventionally used for the protectionof remote carboxy groups which will remain inert during peptidesynthesis. Thus, the ω-carboxy group can be protected in the form of anester, particularly a benzyl ester although other alkyl or aryl estergroups can be used. Other forms of carboxy group protection can be madeprovided that the protected carboxy group remains inert during peptidesynthesis conditions and is readily removable thereafter.

Because the oligopeptides of the invention have a terminal aldehyderesidue, traditional solid phase methods of synthesis are notappropriate and a modification of a reduction method known for thesynthesis of leupeptin is not appropriate in view of the problems ingenerating the terminal aldehyde group by reduction in a compound havingpotentially reducible ω-carboxy groups in the aspartyl residues.Oxidative methods of generating aldehyde groups from the correspondingalcohol have not yet been applied to the synthesis of argininalcontaining peptides so that it was necessary to adopt a new approachbased on fragment condensation. In this method, in addition to theterminal amino and carboxy groups being protected, we also protect theterminal aldehyde group and the quanidinium group if present.

In one of our syntheses, described for the sake of example, for theproduction of a polyaspartyl argininal, a nitro arginine is firstconverted into the corresponding argininal by reduction of the carboxygroup and the aldehyde group is immediately protected by reaction withsemicarbazide hydrochloride to form the semicarbazone. The desiredpolyaspartyl peptide is also constructed, protected at the terminalamino group and the ω-carboxy groups and the polyaspartyl material,finally reacted, using an activated ester synthesis, with theN-unprotected nitroargininal semicarbazone. Finally, the variousprotecting groups are removed to give the polyaspartyl argininal. Thereaction scheme is shown in the following schematic drawing whereterminal amino groups are protected with tertiary butyloxy carbonylgroups, the ω-carboxy groups are protected as benzyl esters and peptidelinkages are formed by reacting N-hydroxysuccinimide esters withN-unprotected amino acids, the terminal carboxy groups beingunprotected. ##STR5##

According to a further feature of the invention, we provide a compoundof formula I as described above insolubilised on a gel matrix, and amethod for the purification of enterokinase comprising bringing asolution of crude enterokinase into contact with such an insolubilisedform of a compound to form an insolubilised conjugate of the compoundand the enterokinase, separating the insolubilised conjugate from thecrude solution and releasing a more pure form of enterokinase from theinsolubilised conjugate.

According to a still further feature of the invention, there is provideda pharmaceutical composition comprising an inert pharmaceuticallyacceptable carrier and, as active ingredient, at least one oligopeptideof formula I as defined above. The oligopeptide I may be formulated, forexample, into enteric coated tablets, emulsions, or solutions for oralor parenteral administration. It may also be linked via its α-aminogroup to a macromolecular carrier such as albumin. Such links may beconstructed with cleavable sites so that the molecule may be cleaved atthe site of action by digestive proteinases. Oligopeptide I may also beincluded in the hypaque solution used during endoscopic retrogradepancreatography--a diagnostic procedure that sometimes inducespancreatitis.

The invention also comprises compounds of formula I for use in a methodof treatment of the human or animal body by surgery, therapy or ofdiagnosis practised on the human or animal body, and in particular, foruse in the alleviation of the symptoms of pancreatitis bychemoprevention or chemotherapy.

The following example is given to illustrate the invention.

A series of peptide aldehydes were prepared by the general methodoutlined in the schematic drawing above, to produce a series ofpolyaspartyl argininals containing 1, 2, 3 or 4 units of aspartic acid.In this example, the following abbreviations are used:

Asp: Aspartic acid

Arg: Arginine

Boc: Tertiarybutyloxycarbonyl

OBzl: Benzyl ester

ONsu: N-hydroxysuccinimide ester

DCC: Dicyclohexylcarbodiimide

HOBt: Tertiary butanol

Z: Benzyloxycarbonyl

TFA: Trifluoroacetic acid

DMF: Dimethylformamide

DIPEA: Diisopropylethylamine

DIBAH: Diisobutylaluminium hydride

TEA: Triethylamine

(a) Boc-Asp(OBzl)-ONSu

19.4 g (60 mmol) Boc-Asp(OBzl) was dissolved in 200 ml dry dioxane,cooled to 4° C. and 7.59 g (66 mmol) N-hydroxysuccinimide followed by13.66 g (66 mmol) DCC was added. The resulting suspension was stirred at4° C. for 24 hours. The precipitate was removed by filtration and washedwith CH₂ Cl₂. The filtrate was rotary evaporated to leave an oil, whichwas taken up in propan-2-ol. Upon leaving in the cold for several dayswhite crystals formed which were filtered, washed with cold propan-2-oland ether, before being vacuum dessicated. Yield 23.3 g (92.5%) mp97°-99° C. (lit 103°-104° C., Laufer and Blout, 1967).

(b) Boc-Asp(OBzl)-Asp(OBzl)

To a suspension of 5.57 g (25 mmol) Asp-(OBzl) in 50 ml 1M NaHCO₃/dioxane (1:1) was added a solution of 10.5 g (25 mmol)Boc-Asp(OBzl)-ONSu, dissolved in 50 ml dioxane. A further 25 ml H₂ O wasadded and the reaction mixture stirred at 4° C. for 2 h. A small amountof solid remained undissolved and the apparent pH (wet pH paper) hadfallen to 6.0. 25 ml 1M NaHCO₃ were added and the reaction mixturestirred for a further six hours by which time the reaction had gone tovirtual completion as monitored by TLC. The reaction products werepoured into 700 ml ice cold water, and cold 2M HCl added to pH 2.5. Theresultant oil was extracted once with 200 ml ethyl acetate and twicewith 250 ml butyl acetate. The combined organic extracts were washedfour times with 100 ml H₂ O. The organic phase was rotary evaporated andthe resultant oil crystallised from toluene-pet ether to yield 8.78 g(66%) mp 88°-91° C. The product comigrated on TLC with that synthesisedpreviously, giving a single ninhydrin positive spot, after exposure toHCl vapour, Rf(B)=0.58, Rf(D)=0.73, Rf(N)=0.43.

(c) Asp(OBzl)-Asp(OBzl). CF₃ COOH

7.92 g (15 mmol) Boc-Asp(OBzl)-Asp(OBzl) was taken up in 100 ml TFA/CH₂Cl₂ (1:1) and stirred at room temperature for 30 minutes. The solventwas rotary evaporated and the residue triturated with ether to give awhite precipitate. Yield 7.49 g (92%). The product gave a singleninhydrin positive spot on TLC Rf(A)=0.75, Rf(B)=0.11, Rf(D)=0.38.

(d) Boc-Asp(OBzl)₃

To 6.5 g (12 mmol) CF₃ COOH. Asp(OBzl)-Asp(OBzl) in 20 ml DMF was added2.06 ml (12 mmol) DIPEA followed by a solution of 5.29 g (12.6 mmol)Boc-Asp(OBzl)-ONSu in 15 ml DMF. After stirring at room temperature forfive hours, TLC analysis showed incomplete removal of the aminocomponent. A further 0.25 g (0.6 mmol) of the active ester was added,and the reaction mixture stirred for a further 3 h; 0.21 ml (1.2 mmol)DIPEA being added for the final 0.5 h. TLC analysis showed completeremoval of the amino component, with a very slight amount of activeester remaining. The reaction mixture was diluted with 250 ml ethylacetate, cooled to 4° C. and washed successively with 2×100 ml 10% (w/v)citric acid, 2×20 ml 5% (w/v) NaHCO₃ and 50 ml 0.2M NaCl. The organicphase was dried over MgSO₄, rotary evaporated and vacuum dessicated.Attempts to crystallise the product were unsuccessful. It gave one majorspot on TLC Rf(B)=0.60, Rf(N)=0.44, Rf(T)=0.14, which was ninhydrinpositive after exposure to HCl with a small quantity of a slower movingimpurity.

(e) [Asp(OBzl)]₃. CF₃ COOH

6.62 g (75% of the oily product from (d) was dissolved in 50 ml TFA/CH₂Cl₂ (1:1) and stirred for twenty minutes. The solvent was rotaryevaporated, the residue triturated with ether and left in the fridgeovernight. The resulting white solid was filtered, washed with coldether and vacuum dessicated over NaOH to yield 6.08 g (8.13 mmol).Single spot on TLC, Rf(A)=0.9, Rf(N)=0.43, Rf(T)=0.10.

(f) Boc-[Asp(OBzl)]₄.

To 5.98 g (8 mmol) [Asp(OBzl)]₃. CF₃ COOH in 20 ml DMF was added 3.70 g(8.8 mmol) Boc-Asp-ONSu followed by 1.38 ml (8.0 mmol) DIPEA. Thereaction mixture was stirred at room temperature for 5 hours, when TLCanalysis showed complete removal of the amino component. The productswere cooled to 4° C., diluted with 250 ml ethyl acetate and washedsequentially with 2×100 ml 10% (w/v) citric acid, 1×50 ml (w/v) NaHCO₃and 50 ml H₂ O. The organic phase was dried over MgSO₄ and rotaryevaporated. The residue was taken up in toluene, pet ether added toslight turbidity and the solution left in the fridge over the weekend togive 4.38 g (4.67 mmol) Boc-[Asp(OBzl)]₄ soft white crystals, mp 76°-79°C., R(f)N=0.45, Rf(T)=0.17, Rf(W)=0.21.

Evaporation of the mother liquor and recrystallisation from toluene/petether gave a second crop 1.71 g, mp 72°-75° C. Total yield 6.1 g (6.5mmol), 81%. Both crops showed two minor slower running impurities onTLC, but were used without further purification.

(g) Z-Arg(NO₂)-semicarbazone

A 250 ml pressure equalising separating funnel was inserted in athree-necked, 500 ml round bottom flask, itself placed in a bath ofacetonitrile/dry ice, with a magnetic stirrer. Nitrogen, dried bypassing over molecular sieve 4A, entered via a hypodermic needleinserted in a rubber septum (Aldrich). 12.4 g (35 mmol) Z-Arg(NO₂) wasvacuum dessicated over P₂ O₅ overnight and then dissolved in 100 ml dryTHF under nitrogen and cooled to 10° C. 6.24 g (38.5 mmol)carbonyldiimidazole was added and the suspension stirred at 10° C. untilall the solids had dissolved. The solution was then cooled to -42° C.100 ml 1M-DIBAH in toluene, (Aldrich), was transferred from the reagentbottle to the separating funnel under nitrogen pressure usingteflon-tubing connected hypodermic needles as described by Lane andKramer (1977). The reducing agent was added dropwise to the stirredreaction mixture over 25 minutes, and stirring continued at =42° C. fora further 30 minutes. The reaction was quenched by the slow addition of250 ml 1.2M-HCl. The resulting suspension was allowed to warm to roomtemperature. 250 ml 0.6M-HCl and 450 ml CHCl₃ were added and theemulsion stirred vigorously for two hours. The organic phase wasseparated from the aqueous phase, and from an oily precipitate whichfailed to partition. The organic extract was washed with water, driedover MgSO₄ and rotary evaporated to give 2.96 g crude aldehyde,Rf(V)=0.5. To this, dissolved in 8 ml 70% (w/v) aqueous ethanol, wasadded 1.39 g (17.4 mmol) sodium acetate and 0.97 g (8.7 mmol)semicarbazide HCl; and the reaction mixture heated at 75° C. until allthe solids had dissolved. The solution was cooled to 4° C. and 10 mlwater added to give a white suspension which was left in the fridgeovernight. The precipitate was centrifuged, washed with cold ethanol andvacuum dessicated to give 1.95 g crude semicarbazone. Recrystallisationfrom absolute ethanol gave white crystals. Yield 1.733 g (3.93 mmol)11.2%. mp 107°-109° C., (lit 107°-109° C., Shimizu et al, 1972).

    ______________________________________                                        Elemental analysis Z--Arg(NO.sub.2)--semicarbazone. C.sub.2 H.sub.5 OH                 C           H      N                                                 ______________________________________                                        Calculated 46.35         6.41   25.45                                         Found      45.24         5.80   27.15                                         ______________________________________                                    

The product gave one spot on TLC which was chlorine-starch/KI and 2, 4,dinitrophenylhydrazine positive, Rf(X)=0.17. (Lit 0.19, Patel andShultz, 1982), with a small amount of an impurity present at the origin.

(h) Arg(NO₂)-semicarbazone

To 1.733 g (3.93 mmol) Z-Arg(NO₂)-semicarbazone, dissolved in 8 mlacetic acid was added 20 ml 45% HBr in acetic acid. After stirring atroom temperature for thirty minutes the solution was poured into 250 mldry ether to yield a yellow precipitate. After leaving in the fridgeovernight, the precipitate was filtered, washed with ether andrecrystallised from methanol. Yield 1.2 g HBr. Arg(NO₂)-semicarbazone.

    ______________________________________                                        Elemental analysis:                                                                         C           H      N                                            ______________________________________                                        Calculated    26.64       5.02   32.85                                        Found         24.27       5.43   31.50                                        ______________________________________                                    

The salt was dissolved in 6 ml water and 0.49 ml (3.52 mmol) TEA added.The solution was left in the fridge overnight to yield a pale greenprecipitate, which was centrifuged, washed with water and ethyl acetateand vacuum dessicated. Yield 545 mg (2.09 mmol). Arg(NO₂)-semicarbazone,mp 178°-180° C. decomposed (lit decomposed at 186°-188° C., Shimizu etal, 1972).

    ______________________________________                                        Elemental analysis                                                                          C           H      N                                            ______________________________________                                        Calculated    32.30       6.20   43.06                                        Found         32.26       6.18   42.11                                        ______________________________________                                    

The product gave a single ninhydrin positive spot on TLC and HVPE at pH6.5 Rf(O)=0.40, Rf(P)=0.52, R(Asp)=+0.52 (predicted +0.64).

(i) Boc-[Asp(OBzl)]_(n) -Arg(NO₂)-semicarbazone (n=1, 2, 3, 4)

Boc-[Asp(OBzl)]₂, Boc-[Asp(OBzl)]₃ and Boc-[Asp(OBzl)]₄, 0.525 L mmol ofeach dissolved in 1 ml DMF, were activated at 4° C. by adding 83 mg(0.575 mmol) HOBt and 120 mg (0.575 mmol) DCC. The resulting suspensionswere stirred for 40 minutes at room temperature, filtered and the solidwashed with 1 ml DMF. To each of the three filtrates, and to a solutionof 0.525 mmol Boc-Asp(OBzl)ONSu in 1 ml DMF, all at 4° C., was added 130mg (0.5 mmol) aliquots of Arg(NO₂)-semicarbazone. The solutions werestirred at 4° C. for 7 hours. A further 12 mg DCC was added to eachreaction vessel, and stirring continued at 4° C. for a further 15 hourswhen TLC analysis showed removal of the amino component. The solutionswere filtered, the solid washed with 1 ml DMF and 20 ml of water addedto each filtrate to yield oily precipitates, which were vacuumdessicated over P₂ O₅. The resulting solids were recrystallised frompropan-2-ol (for n=1) or triturated with EtOAc, a little diethyl etheradded and left in the fridge overnight, (for n=2, 3, 4). The solids werecentrifuged, washed with cold EtOAc and vacuum dessicated.

    ______________________________________                                        Yields                                                                        ______________________________________                                        Boc--Asp(OBzl)--Arg(NO.sub.2)--semicarbazone                                                            142 mg (50%)                                        Rf(T) = 0.20 Rf(W) = 0.06                                                     Boc--[Asp(OBzl)].sub.2 --Arg(NO.sub.2)--semicarbazone                                                   253 mg (65%)                                        Rf(T) = 0.36 Rf(W) = 0.13                                                     Boc--[Asp(OBzl)].sub.3 --Arg(NO.sub.2)--semicarbazone                                                   270 mg (55%)                                        Rf(T) = 0.43 Rf(W) = 0.16                                                     Boc--[Asp(OBzl)].sub.4 --Arg(NO.sub.2)--semicarbazone                                                   318 mg (54%)                                        Rf(T) = 0.50 Rf(W) = 0.19                                                     ______________________________________                                    

The products gave one ninhydrin, fluorescent and 2, 4dinitrophenylhydrazine positive spot on TLC with the mobilities givenabove. Boc-Asp(OBzl)-Arg(NO₂)-carbazone showed a minor impurity at theorigin.

(j) Boc-[Asp(OBzl)]_(n) -(NO₂)-Argininal (n=1, 2, 3, 4)

To 0.15 mmol of the four semicarbazones, dissolved in 1.5 ml MeOH wasadded 1.5 mmol formaldehyde (0.122 ml 37% (v/v) in H₂ O) and 1.5 mmol(0.086 ml) glacial acetic acid. For n=3 and n=4 a small precipitateformed which was solubilised by addition of 0.25 ml and 0.5 ml DMFrespectively. The solutions were stirred at room temperature; furtheraliquots of 62 μl formaldehyde and 42 μl glacial acetic acid being addedafter 2 hours (for n=1, 2, 3, 4) and 7 hours (for n=3, 4). After 24hours TLC analysis showed complete conversion of the starting materialsto faster running components. Approximately 5 ml H₂ O was added to eachsolution to yield white suspensions. After cooling in the fridgeovernight the suspensions were centrifuged, and the pellets washed twicewith water before being dessicated.

    ______________________________________                                        Yields                                                                        ______________________________________                                        Boc--Asp(OBzl)--(NO.sub.2)Argininal                                                                    25 mg                                                Rf(B) = 0.51 Rf(T) = 0.57 Rf(W) = 0.21                                        Boc--[Asp(OBzl)].sub.2 --(NO.sub.2)Argininal                                                          103 mg                                                Rf(B) = 0.62 Rf(T) = 0.63 Rf(W) = 0.23                                        Boc--[Asp(OBzl)].sub.3 --(NO.sub.2)Argininal                                                          140 mg                                                Rf(B) = 0.66 Rf(T) = 0.67 Rf(W) = 0.24                                        Boc--[Asp(OBzl)].sub.4 --(NO.sub.2)Argininal                                                          169 mg                                                Rf(B) = 0.71 Rf(T) = 0.71 Rf(W) = 0.26                                        ______________________________________                                    

The products each gave one ninhydrin (after exposure to HCl vapour),fluorescent and 2, 4 dinitrophenylhydrazine positive spot on TLV withthe mobilities given above. Trace impurities at Rf(T)=0.0 and 0.21 werevisible in all samples.

(k) Boc-(Asp)_(n) -Argininal (n=1, 2, 3, 4)

The fully protected peptide aldehydes were dissolved in 1.5 ml MeOH towhich was added 30 μl glacial acetic acid and 250 μl H₂ O. The sidechain protecting groups were removed by catalytic hydrogenation over 20mg Pd/charcoal. After 24 hours the catalyst was removed by filtration,the filtrate freezed dried and vacuum dessicated over P₂ O₅.

    ______________________________________                                                          % peptide                                                               Yield by weight.sup.a                                                                          Asp:Argininal.sup.b                              ______________________________________                                        Boc--Asp--Argininal                                                                         19 mg   70%        1.2:1                                        Boc--(Asp).sub.2 --Argininal                                                                51 mg   98%        3.4:1                                        Boc--(Asp).sub.3 --Argininal                                                                76 mg   89%        4.6:1                                        Boc--(Asp).sub.4 --Argininal                                                                81 mg   96%        6.9:1                                        ______________________________________                                         .sup.a based on the aspartic acid ccntent as determined by amino acid         analysis                                                                      .sup.b argininal estimated by quantitative Sakaguchi reaction            

Alternate synthetic procedures have also been employed. LiAlH₄ has beenused to reduce CbZ- or Boc-(NO₂) L arginine imidazolide and theprotecting group removed by treatment with saturated HCl in dioxane. Theprotected tetraaspartyl peptide mediated DCC coupling instead of withhydroxysuccinimide esters linking Boc(Asp OBZ)₂ to (Asp OBZ)₂. Removalof O benzyl groups on the aspartyl side chains, and the NO₂ -protectinggroup on the guanidine moiety has been achieved by catalytic transferhydrogenation from ammonium formate in the presence of PdC (5%).

BIOLOGICAL RESULTS

Biological testing was carried out on the polyaspartyl argininalmaterials prepared as described above without further purification. Inorder to demonstrate their selective inhibitory action towardsenterokinase, comparative tests were carried out between the materialsof the invention and leupeptin. Inhibition constants K_(i) for human andpig enterokinase were calculated using α-N-[³ H] acetyl trypsinogen andGly-(Asp)₄ -Lys-2-Nap as substrates using linear regression analysis ofl/v against [I] at three substrate concentrations. Inhibition of trypsincatalysed hydrolysis of both these substrates and Bz-Arg-OEt wasexpressed as the IC₅₀, the concentration of inhibitor required to reducetrypsin activity by 50%. The results to activation peptides, fourpeptides of the invention, leupeptin and p-aminobenzamidine are set outin Tables 1 to 3 below.

                  TABLE 1                                                         ______________________________________                                        INHIBITION CONSTANTS FOR HUMAN                                                ENTEROKINASE (K.sub.i) AND TRYPSIN                                            (IC.sub.50) WITH Gly--(Asp).sub.4 --Lys--2-Nap AS SUBSTRATE                                 Substrate                                                                     Gly--(Asp).sub.4 --Lys--2--Nap                                                  Human         Trypsin                                         Inhibitor       Enterokinase (K.sub.i)                                                                      (IC.sub.50)                                     ______________________________________                                        Boc(Asp).sub.1 Argal                                                                          80 ± 5 μM                                                                             >500 μM                                      Boc(Asp).sub.2 Argal                                                                           0.2 ± 0.05 μM                                                                        ˜20 μM                                 Boc(Asp).sub.3 Argal                                                                           0.1 μM    ˜50 μM                                 Boc(Asp).sub.4 Argal                                                                          0.055 ± 0.005 μM                                                                      ˜50 μM                                 Leupeptin        1.0 μM    0.175 μM                                     (Asp).sub.4 Lys 100 μM     --                                              Ala--Pro--Phe--(Asp).sub.4 Lys                                                                750 μM     --                                              ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    INHIBITION CONSTANTS FOR HUMAN                                                ENTEROKINASE (K.sub.i), PIG ENTEROKINASE (K.sub.i) AND                        TRYPSIN (IC.sub.50) WITH α-N--[.sup.3 H] ACETYL TRYPSINOGEN AS          SUBSTRATE                                                                               Substrate                                                                     α-N--[.sup.3 H]acetyltrypsinogen                              Inhibitor Human Enterokinase (K.sub.i)                                                               Pig Enterokinase (K.sub.i)                                                               Trypsin (IC.sub.50)                         __________________________________________________________________________    Boc(Asp).sub.1 Argal                                                                    50 ± 25 μM                                                                           55 ± 10 μM                                                                         >300 μM                                  Boc(Asp).sub.2 Argal                                                                    0.01 ± 0.005 μM                                                                      0.05 ± 0.01 μM                                                                      >30 μM                                  Boc(Asp).sub.3 Argal                                                                    0.02 ± 0.005 μM                                                                      0.035 ± 0.005 μM                                                                   >100 μM                                  Boc(Asp).sub.4 Argal                                                                    0.01 ± 0.005 μM                                                                      0.02 ± 0.01 μM                                                                     >100 μM                                  Leupeptin   1.0 μM  ≧1.0 μM                                                                          0.35 μM                                p-aminobenzamidine                                                                      ≧50 μM                                                                            ≧50 μM                                                                        ≦300 μM                           __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    INHIBITION CONSTANTS FOR TRYPSIN (IC.sub.50) WITH Bz--Arg--OEt AS             SUBSTRATE                                                                             Substrate                                                                     Trypsin (IC.sub.50)                                                           Boc(Asp).sub.n Argal                                                  Inhibitor                                                                             1  2     3     4     Leupeptin                                                                            p-aminobenzamidine                                                                      (Asp).sub.4 Lys                 __________________________________________________________________________    Bz--Arg--OEt                                                                          N.D.                                                                             >500 μM                                                                          >750 μM                                                                          >750 μM                                                                          2.5 ± 0.5 μM                                                                   ≦150 μM                                                                       >1500 μM                     __________________________________________________________________________

The results show the specific inhibitory activity of the inventionoligopeptides with respect to enterokinase, particularly of thetetraaspartyl argininal material.

An example of the use of compounds described in formula I as α-NH₂immobilised ligands for the affinity chromatography of mammalianenterokinase is given as follows: Boc(Asp OBZ)₄ argininal semicarbazonewas treated with 4M HCl/dioxane for 30 min., and evaporated. Theprecipitated hydrochloride was filtered off, washed with dioxane, andhydrogenated in methanol with Pd/C (5%) for 12 h. at room temperature.The catalyst was filtered off, the deprotected semicarbazone isolated,and incubated (10 mg/μl) with Affi-gel 10 (Bio-Rad, Richmond, Ca., USA)in 0.1M Hepes-NaOH, pH 7.4, containing 10 mM CaCl₂ for several hours at4° C., followed by capping with 1M glycine (pH 8). The aldehyde wasregenerated on the affinity matrix by treatment with excess formaldehydein methanol with 5% acetic acid for 12 h. at room temperature. Theaffinity matrix was washed with 1M NaCl, and equilibrated in 0.05Hepes-NaOH (pH 7.6), containing 10 mM CaCl₂, and a sample ofenterokinase-containing biological fluid applied. The purified enzymewas eluted by lowering the pH of the operational buffer and the additionof EDTA.

We claim:
 1. A compound of the general formula: ##STR6## wherein n is aninteger from 2 to 6, m is an integer from 2 to 4, p is an integer from 1to 3, R is H, an amino protecting group conventionally used in peptidechemistry or a solid phase support, R₁ is H or a carboxy protectinggroup conventionally used in peptide chemistry and R₂ is H or alkyl or##STR7##
 2. A compound according to claim 1 wherein m is 3 and R₂ is##STR8##
 3. A compound according to claim 1 wherein m is 4 and R₂ isCH₃.
 4. A compound according to any one of the preceding claims whereinp is 1 and R₁ is H.
 5. A compound according to any one of the precedingclaims wherein n is 2 or
 4. 6. A compound according to any one of thepreceding claims wherein R is tertiary butyloxycarbonyl or acetyl.
 7. Acompound according to claim 1 of the formula: ##STR9## in which n is 2or
 4. 8. A compound according to any one of the preceding claimsinsolubilised on a gel matrix.
 9. A pharmaceutical composition foralleviating the symptoms of pancreatitis comprising an effective amountof a compound according to any one of claims 1 to 7 together with aninert pharmaceutically acceptable carrier or diluent.
 10. A method oftreatment for alleviating the symptoms of pancreatitis comprisingadministering to a host in need of such treatment an effective amount ofa compound according to any one of claims 1 to 7.